Abstract. Signaling and Hypertrophy in Aged Rat Skeletal Muscle. By: Hoke Whitworth. November, Director of Thesis: Dr. Scott E.

Transcription

1 Abstract Paired Effects of Dietary Leucine Supplementation and Overload on Protein Translational Signaling and Hypertrophy in Aged Rat Skeletal Muscle By: Hoke Whitworth November, 2012 Director of Thesis: Dr. Scott E. Gordon Department of Kinesiology Sarcopenia is an age-associated disorder that causes loss of skeletal muscle mass, particularly in type II (fast-twitch) muscle fibers. This loss in muscle mass can cause disability, reductions in the quality of life, and can contribute to the development of other more lifethreatening morbidities and even death. Researchers have utilized muscle overloading and ergogenic aids, such as whey protein and essential amino acids (specifically leucine), in rats and humans in attempts to reduce or attenuate these losses as part of a primary prevention strategy. Unfortunately, there is also a loss of overload-induced growth capacity in aged fast-twitch skeletal muscle. However, no studies have explored the potential synergistic effect of leucine supplementation on overload-induced skeletal muscle growth in aged animals. To that end, the purpose of this study was to examine the effects of dietary leucine supplementation on protein translational signaling and hypertrophy in the overloaded fast-twitch skeletal muscles of aged animals. It was hypothesized that supplementing a standard chow diet with 5% leucine would enhance muscle hypertrophy in overloaded fast-twitch plantaris muscles of aged (33-month old) rats to levels observed in young adult (8-month old) rats. It was also hypothesized that 5% dietary leucine supplementation would enhance protein translational [70 kda ribosomal protein S6 kinase (p70s6k), ribosomal protein S6 (rps6), eukaryotic elongation factor 2 kinase (eef2k),

2 and eef2] signaling in the overloaded fast-twitch plantaris muscles of aged rats to levels observed in young adult rats. Young adult and old male Fisher 344 x Brown Norway F1 Hybrid (FBN) rats underwent a 1-week unilateral plantaris muscle overload via tenotomy of the synergistic gastrocnemius muscle. Within each age group, animals were matched for body weight and separated into either a dietary leucine supplementation group (additional 5% leucine content in place of 5% of the carbohydrate content in normal rat chow starting 2 days prior to, and throughout, the overload intervention; n = 7/age group) or placebo group (normal rat chow; n = 6/age group). No differences in daily calorie consumption were observed between the placebo vs. leucine groups within each age group. Plantaris muscles were harvested at the end of the overload period. Dietary leucine enrichment significantly (p 0.05) enhanced overload-induced fast-twitch plantaris muscle hypertrophy in old, but not in young adult, animals. Additionally, western blotting analyses revealed that phospho-p70s6k (Thr389) and phospho-rps6 (Ser235/Ser236) were significantly lower in old vs. young overloaded muscles under placebo conditions, but leucine partially restored both phospho-p70s6k and phospho-rps6 in old overloaded muscles to that of young adult overloaded muscles. Overload significantly increased eef2k phosphorylation in young, but not in old animals, and leucine supplementation had no affect on eef2k phosphorylation in either age group. Overload significantly increased total eef2 content and decreased inhibitory eef2 phosphorylation (Thr56; normalized to total eef2) in young adult muscles regardless of leucine supplementation. Total eef2 content was unaffected by overload in old placebo muscles, but leucine supplementation in old animals nonsignificantly (p = 0.09) restored the overload-induced increase in total eef2 content. These novel findings indicate that a leucine-enriched diet may potentially enhance overload-induced

3 growth of aged fast-twitch muscle, in part by enhancing pathways known to stimulate protein synthesis.

4 Paired Effects of Dietary Leucine Supplementation and Overload on Protein Translational Signaling and Hypertrophy in Aged Rat Skeletal Muscle A Thesis Presented to the Faculty of the Department of Kinesiology In Partial Fulfillment of the Requirements for the Degree Master of Science in Exercise Physiology By Hoke B. Whitworth, Jr. November, 2012

5 Hoke B. Whitworth, Jr., 2012

6 Paired Effects of Dietary Leucine Supplementation and Overload on Protein Translational Signaling and Hypertrophy in Aged Rat Skeletal Muscle by Hoke B. Whitworth, Jr. Approved by: Director of Dissertation/Thesis: Dr. Scott E. Gordon, PhD Committee Member: Dr. Peter A. Farrell, PhD Committee Member: Dr. Carol A. Witczak, PhD Committee Member: Dr. Terry E. Jones, PhD Chair of the Department of Kinesiology: Dr. Stacey R. Altman, JD Dean of the Graduate School: Dr. Paul Gemperline, PhD

7 Dedications I could not have completed this literary work without the unwavering support of my friends and family. I am forever grateful. As a token of my gratitude, I wish to dedicate this work to you all.

8 Acknowledgements I would like to thank Dr. Scott Gordon for his mentoring and contributions to this project. Along with my mentor, I would like to thank the members of my thesis committee, Dr. Peter Farrell, Dr. Carol Witczak, and Dr. Terry Jones, for their service and advising throughout this process. I would also like to recognize my counterpart, William Mixon, on this project, coinvestigator Dr. Rengfei Shi, and research assistant Clay Myers. The current work could not have been completed without you. Lastly, I would like to thank my family and friends for your support throughout this academic endeavor.

9 Table Of Contents List of Tables.pg xi List of Figures...pg xii Chapter I: Introduction.pg 1 Sarcopenia.pg 1 Protein Synthesis...pg 2 Aging and Protein Synthesis.pg 3 Muscle Overloading and Protein Synthesis..pg 4 Supplementation and Protein Synthesis pg 5 Aims.. pg 5 Hypothesis.pg 6 Chapter II: Review of Literature..pg 7 Muscle Protein Initiation and Elongation Signaling.....pg 8 Muscle Overloading and Resistance Exercise.. pg 13 Supplementation of Whey Protein and Amino Acids...pg 20 Nutritional Supplementation Paired with Muscle Overload and Resistance Exercise.pg 30 Chapter III: Methods....pg 40 Experimental Animals......pg 40 Rationale for Experimental Animals....pg 40 Dietary Intervention..pg 41 Synergist Tenotomy Procedure.....pg 43 Tissue Harvesting and Euthanasia....pg 43

10 Western Blot Analysis.. pg 44 Statistical Analysis... pg 46 Chapter IV: Results.pg 47 Body Weight.pg 47 Food Intake...pg 48 Muscle Wet Weight/Hypertrophy.....pg 49 Protein Content.pg 52 p70s6k Western Blotting Analysis...pg 52 rps6 Western Blotting Analysis pg 58 eef2k Western Blotting Analysis.pg 64 eef2 Western Blotting Analysis......pg 70 Chapter V: Discussion.pg 76 Conclusion pg 83 Practical Applications... pg 83 Chapter VI: References....pg 85 Appendix A: ECU Animal Use Protocol Approval Document... pg 93

11 List of Tables Table 3.1 pg 42 Table 4.1 pg 47 Table 4.2 pg 48

12 List of Figures Figure 2.1..pg 9 Figure 4.1..pg 50 Figure 4.2. pg 51 Figure 4.3..pg 53 Figure pg 55 Figure 4.5..pg 57 Figure pg 59 Figure pg 61 Figure pg 63 Figure 4.9..pg 65 Figure 4.10 pg 67 Figure 4.11 pg 69 Figure 4.12 pg 71 Figure 4.13 pg 73 Figure 4.14 pg 75

13 1 Chapter I: Introduction Sarcopenia As humans age, one of the many physiological processes that leads to disease development and disability is the loss of skeletal muscle. This age-associated decline in skeletal muscle is sarcopenia (Evans, 1995). Sarcopenia has been linked to a reduction in muscle fiber size and number (Welle, 2002), particularly in type II, fast-twitch fibers (reviewed in Lexell, 1995). Muscle fiber degeneration takes place primarily in type II muscle fiber size, with very little, if any, reduction in type I fiber size (reviewed in Lexell, 1995, Thomson and Gordon, 2006). Animal models have demonstrated reductions in total muscle mass and cross sectional area, as well as, declines in individual muscle fiber cross sectional area (Blough, 2000). A decrease in muscle mass has a positive correlation with decreases in strength (Frontera, 2000) and contractile quality (Blough, 2000). This loss in muscle quality generates functional limitations (Hairi, 2010) and physical disability (Dorrens, 2003), specifically in normal activities of daily living among elderly individuals (Dorrens, 2003; Hairi, 2010). An increased risk of falling among elderly individuals has also been correlated with excessive losses in muscle strength (Wickham, 1989). An estimated ~1.5% of the total national healthcare expenditure in the US has been directly attributed to sarcopenia. This seemingly minuscule percentage equates to an approximate monetary value of 18.5 billion US dollars (Janssen, 2004). Although this chronic process has been defined, it is difficult to diagnose sarcopenia because no absolute values have been established for losses of fat free mass, cell mass, or total muscle mass (Roubenoff, 2000). Muscle fiber degeneration can be seen in both genders, although it can affect males and females at different rates and times throughout their middle and late adult lives. Males lose an

14 2 estimated 1.9 kg/decade, while females are slightly less affected with losses of 1.1 kg/decade (Janssen, 2000). Over the span of a human life, losses in muscle area up to 40% have been documented in participants between 20 and 80 years of age (reviewed in Lexell, 1995). Lean body mass can play a role in the survival rate for individuals suffering from lifethreatening illnesses (Tellado, 1988). There is a strong correlation between muscle mass and strength. As stated previously, this loss in strength can become debilitating even for carrying out normal activities of daily living. However, a loss of muscle mass is not always accompanied by a reduction in strength (Roubenoff, 2000). In order to determine what causes the loss of muscle fibers with age, a closer examination must be performed on muscle protein synthesis and degradation pathways at the molecular level. Protein Synthesis Growth within, and maintenance of, existing muscle fibers is a complex process incorporating several different pathways. The muscle protein synthesis translational pathway consists of three separate stages (initiation, elongation, and termination) that work together to form specific proteins corresponding to the blueprint laid out by DNA in the cell (Nadar, 2002; Wang, 2006). The main areas of focus for this investigation were protein translation initiation and elongation. One pathway that has been recognized for stimulating protein synthesis is the mammalian target of rapamycin (mtor) pathway (Figure 2.1). Cell size regulation has been attributed partly to this pathway (Bodine, 2001). Activation and deactivation of the specific signaling proteins depends on phosphorylation or dephosphorylation status. The protein mtor controls two important downstream signaling proteins involved in the initiation process. Eukaryotic initiation factor 4E binding protein (4E-BP1) acts to inhibit protein translational signaling by

15 3 binding with eif4e. When phosphorylated by mtor, 4E-BP1 loses its affinity for eif4e, allowing eif4e to participate in the formation of the ribosomal complex eif4f. The second signaling protein controlled by mtor is p70 ribosomal protein S6 kinase, or p70s6k. P70s6k acts to phosphorylate the S6 subunit of the 40S ribosome involved in the translation initiation (Nadar, 2002). The second step in this translational pathway involves elongation of the protein chain. Eukaryotic elongation factor 2 (eef2) is one of two important factors required for this process to successfully occur (Wang, 2006). EEF2 can only participate in the elongation process when it has been dephosphorylated. Activation of eef2 is controlled by its respective kinase, eef2k. Phosphorylation of eef2k allows for eef2 to become active. Researchers have postulated that p70s6k has the ability to phosphorylate eef2k on one of its inhibitory sites. This could be indicative of a relationship between signaling proteins found in translation initiation and elongation pathways (Fick, 2007). Aging and Protein Synthesis Aging may inhibit certain mechanisms within the pathways described above and create a decline in protein synthesis (Welle, 1993; Welle, 1994; Parkington 2004; Bagalopal, 1997; Paturi, 2010). This lower protein synthesis, particularly in type II muscle fibers, is believed to be one of the main contributors for the development of sarcopenia in the elderly (Parkington, 2004; Thomson and Gordon, 2005; Thomson and Gordon, 2006; Paturi, 2010). Moreover, normal stimulation of muscle protein synthesis with muscle overloading declines with age making it difficult for individuals to counteract the effects of sarcopenia (Blough, 2000; Thomson and Gordon, 2005; Thomson and Gordon, 2006). Our investigation will analyze signaling proteins (located downstream from mtor) involved in the Akt-mTOR pathway (p70s6k, rps6, eef2k

16 4 and eef2). An assessment of phosphorylation and abundance status of these signaling proteins, and changes in hypertrophy, will be performed to evaluate the effectiveness of possible intervention strategies (chronic muscle overload and leucine supplementation) used to attenuate sarcopenia. Muscle Overloading and Protein Synthesis Overloading skeletal muscle with an external force has been shown to activate skeletal muscle protein synthesis and enhance the development of mixed muscle and myofibrillar proteins (Yarasheski, 1999; Hasten, 2000). Translational proteins demonstrate increased phosphorylation/activation in the initiation stage, particularly with mtor, 4E-BP1, p70s6k, and rps6, and dephosphorylation/activation of eef2k and eef2 in the elongation stage (Cuthbertson, 2005; Parkington, 2004; Thomson and Gordon, 2006; Kumar, 2009). Increases in muscle cross sectional area, lean muscle mass, and strength are also positive outcomes of chronic muscle overload (Parkington, 2004). Suppression of hypertrophic responses to chronic resistance training has been demonstrated in aging rats (Blough, 2000; Thomson and Gordon, 2005; Thomson and Gordon, 2006) and humans (Kumar, 2009). Also, measurably smaller activations of mtor, 4E-BP1, and p70s6k1 have been observed in older muscle when compared to muscles in younger counterparts. Yet these increases do still occur in response to resistance training (Kumar, 2009; Cuthbertson, 2005). It has been suggested that increasing the amount of mechanical overload in skeletal muscle could be used as an intervention to delay, attenuate, or perhaps even reserve the effects of sarcopenia (Kosek, 2006). The effects of muscle overloading on muscle hypertrophy and protein translational signaling in aging muscle will be evaluated in the current study. The effects on aged muscle will be compared to the responses found in adult rats.

17 5 Supplementation and Protein Synthesis Researchers believe that amino acid availability is an important factor for protein synthesis (Hara, 1998; Fujita, 2007). Various ergogenic aids, such as whey protein, essential amino acids, and leucine, have been supplemented to determine if enhancements in muscle protein synthesis can be made above that of normal homeostatic responses to overload. The addition of essential amino acids (EAA) to a normal diet has generated improvements in skeletal muscle protein synthesis (Katsanos, 2008; Paddon-Jones, 2006). Signaling within the synthesis pathway causes mtor and certain downstream components of translation initiation and elongation (mentioned previously) to become activated (Hara, 1998; Crozier, 2005; Du, 2007). Chronic supplementation of EAA also enhances lean body mass and basal protein synthesis. Specifically, leucine, one key essential amino acid, has proven to alter phosphorylation and activation status of mtor (Suryawan, 2008). Whole body protein degradation also becomes suppressed with the supplementation of leucine (Koopman, 2006 #2). Leucine supplementation has exhibited an enhanced effect on muscle protein signaling, specifically in the Akt-mTOR pathway, and fractional protein synthesis rates in the muscles of elderly individuals. Increases in the amount of leucine administered to aged participants have generated positive gains in protein signaling and muscle protein synthesis reaching similar values observed in young participants (Katsanos, 2006). Using this information, we postulate that the addition of leucine to a normal diet will increase the stimulation of muscle protein synthesis in the overloaded muscles of rats. Aims To the best of our knowledge, there have been no studies observing the combined effects of leucine supplementation and muscle overloading on translational signaling and protein

18 6 synthesis in elderly rodents or human beings. Therefore, the purpose of this study was to examine the effects of dietary leucine supplementation on protein translational signaling and hypertrophy in the overloaded fast-twitch skeletal muscles of aged animals. Hypothesis It is hypothesized that supplementing a standard chow diet with 5% leucine will enhance muscle hypertrophy in overloaded fast-twitch plantaris muscles of aged (33-month old) rats to levels observed in young adult (8-month old) rats. It is also hypothesized that 5% dietary leucine supplementation will enhance p70s6k, rps6, eef2k, and eef2 signaling in the overloaded fasttwitch plantaris muscles of aged rats to levels observed in young adult rats. If this intervention proves to be successful, it will support the possibility of supplementing leucine during chronic resistance training to enhance gains in muscle mass and strength in elderly humans. This could lead to an improvement in quality of life with age and a decline in progressive ailments associated with age.

19 7 Chapter II: Review of Literature Sarcopenia is a debilitating disorder caused by an age-associated loss of skeletal muscle (Evans, 1995), primarily in type II muscle fibers (reviewed in Lexell, 1995). Losses in skeletal muscle area reach values as high as 40% for individuals ranging between 20 to 80 years of age (reviewed in Lexell, 1995). Reductions in muscle strength (Frontera, 2000) and contraction quality (Blough, 2000) accompany this disorder and can lead to a reduced ability to function (Dorrens, 2003; Hairi, 2010). Furthermore, a decline in skeletal muscle can lead to a reduced survival rate among individuals suffering from life-threatening illness (Tellado, 1988) Skeletal muscle fiber atrophy has been attributed to an imbalance in the ratio of muscle protein synthesis to protein degradation (Kimball, 2010). Recognized methods of stimulating hypertrophy, such as muscle overloading, have demonstrated a limited ability to increase protein translational signaling and muscle mass with age (Blough, 2000; Thomson and Gordon, 2005; Chale-Rush, 2009). The signaling protein mtor and downstream signaling markers (p70s6k, rps6, eef2k, and eef2), which are involved in muscle protein translation, have been correlated with the reduced ability to stimulate muscle hypertrophy with age (Thomson and Gordon, 2005; Chale-Rush, 2009). Leucine, a branched-chain amino acid, has shown promising effects on protein anabolism and translational signaling marker phosphorylation enhancement among young adult rats and humans when combined with EAA (essential amino acids) and other nutrients (Anthony, 2000; Fujita, 2007) or administered independently (Anthony, 2000 #2; Anthony, 2002 #2; Crozier, 2005 Suryawan, 2008). Enhancements in muscle protein synthesis and translational signaling have also been observed in old rats and humans when dosages of amino acids are increased (Dardevet, 2002; Paddon-Jones, 2006). In the current study, we chose to analyze the combined

20 8 effects of dietary leucine-enrichment and muscle overloading in an attempt to discover a possible intervention for reversing the age-associated reduction in muscle hypertrophy and muscle translational signaling. To our knowledge, the combined effects of a leucine-enriched diet and chronic muscle overload on muscle hypertrophy and muscle protein translation in old rats have yet to be measured. Muscle Protein Translation Initiation and Elongation Signaling One particular protein that plays a significant role in translational signaling is mammalian target of rapamycin (mtor). In addition to its involvement in protein translation, mtor has a multitude of functional roles including participation in cell proliferation, apoptosis, and autophagy, (reviewed in Miyazaki, 2009). mtor is comprised of two distinct isoforms: mtorc1 and mtorc2. A specific binding partner has been identified for each isoform: raptor and rictor, respectively (Wang, 2006). Rapamycin is a specific pharmaceutical inhibitor of raptor binding mtorc1 and, when administered, almost completely prevents any in vivo muscle hypertrophic response to overload (Bodine, 2001). Rapamycin has been utilized to determine whether certain stimuli could control mtor signaling (Bodine, 2001; Ionki, 2003; Ionki, 2003 #2) and which downstream signaling proteins from mtor were affected (Redpath, 1996; Wang, 2001; Fingar, 2002; Browne, 2004).

21 9 Figure 2.1: mtor and signaling proteins. One specific pathway that leads to the activation of mtor and its downstream signaling proteins is the insulin growth factor-1 (IGF-1) pathway (Figure 2.1). Hormones or growth factors, such as insulin or IGF-1, have been shown to stimulate this pathway. Once activated, phosphoinositide-3-kinase (PI(3)k) phosphorylates and activates Akt (also known as protein kinase B (PKB)) (Rommel, 2001). Akt controls a direct mtor-inhibiting complex known as tuberous sclerosis protein 1 and 2 (TSC1/TSC2) (Ionki, 2003). From there, TSC1/TSC2 facilitates the hydrolysis of GTP to GDP on Rheb and allows for activation of mtor (Ionki, 2003 #2). The increased availability of nutrients, such as amino acids, has also been associated with increased phosphorylation of signaling proteins (4E-BP1 and p70s6k) controlled by mtor (Anthony, 2000; Anthony, 2000 #2; Anthony, 2002 #2; Fujita, 2007; Atherton, 2010). However, nutrient signaling is believed to occur through separate pathways than those controlled by

22 10 hormones or growth factors (Hara, 1998; Drummond, 2010). Specific upstream complexes known as Rag A/B and Rag C/D GTPases are believed to contribute to the altered phosphorylation status of 70 kda ribosomal protein S6 kinase (p70s6k) after amino acids are administered (Kim, 2008). Cellular amino acid transporters LAT1/CD98 (L-type amino acid transporter with and a glycoprotein) and SNAT2 (sodium-coupled neutral amino acid transporter) have also demonstrated an involvement in mrna expression and mtor activity (Drummond, 2010). Additionally, human vacuolar protein sorting 34 (hvps34) increased phosphorylation of proteins downstream from mtor (rps6 and p70s6k1), without having any effect on proteins immediately upstream (TSC2, PKB/Akt), after amino acids were administered (Nobukuni, 2005). As mentioned previously, mtor controls the activity of two signaling proteins involved in the translational initiation and elongation stages of protein synthesis (Figure 2.1). The first of which is eukaryotic initiation factor 4E binding protein 1 (4E-BP1). This protein plays a pivotal role in the formation of the eif4f complex (composed of eif4e, eif4g, and eif4a) by either preventing or allowing eif4e to interact with the other two components. mtor phosphorylates 4E-BP1 and releases the inhibitory bond between 4E-BP1 and eif4e (reviewed in Kimball, 2010). Although this protein and the processes involved in protein translation initiation are important in synthesizing new proteins, we have chosen to focus our attention on the other protein that is regulated by mtor; ribosomal protein S6 kinase p70 (p70s6k) (Nadar, 2002). mtor s control of p70s6k has been observed in vivo (Sprague-Dawley rats) and in vitro (C2C12 myoblasts). Following a treatment of rapamycin, in vivo increases in the phosphorylation of p70s6k in response to exercise (Bodine, 2001), as well as in vitro in C2C12 myoblasts (Rommel, 2001) were attenuated.

23 11 P70s6k is phosphorylated and activated by mtor on residue Thr389 (Nadar, 2002). Although p70s6k controls many aspects of cellular activity (reviewed in Magnuson, 2012), our focus is specifically on the two components p70s6k regulates for cellular growth: ribosomal protein S6, rps6 (Ruvinsky, 2005), and eukaryotic elongation factor 2 kinase, eef2k (Browne, 2002). Similar to mtor, increases in phosphorylation of p70s6k occur in vivo (Parkington, 2004; Thomson and Gordon, 2005) in the overloaded muscles of rats. Increases in p70s6k phosphorylation were also observed following an acute bout(s) of resistance training in humans (Eliasson, 2006; Dreyer, 2006; Dreyer, 2010; Terzis, 2010) and increases in response to resistance training stimuli were dose-dependent (Terzis, 2010). mtor has facilitated the phosphorylation of a downstream signaling protein, 40S ribosomal protein S6 through p70s6k (Nadar, 2002; Wang, 2006). The exact roles of ribosomal protein S6 are still unclear. One function that is still up for debate is the possibility that rps6 assists in upregulating 5 -terminal oligopyrimidine (5 -TOP) sequences, which participate in coding for translational machinery (Jefferies, 1997; Magnuson, 2005). However, it has been postulated that p70s6k-rps6 pathway is not the only means of upregulating 5 TOP mrna (Jefferies, 1997; Ruvinsky, 2005). Regardless, it is clear that rps6 does participate in the regulation of cell size (Ruvinsky, 2005), which is why rps6 was chosen as one of the signaling markers of protein translation to analyze in this study. The intermediate stage of muscle protein translation is elongation. This process involves two eukaryotic elongation factors: eef1 and eef2 (reviewed in Wang, 2006). The elongation process actually generates the bulk of the protein chain. eef2 is a monomer and will be the focus of this study. eef2 is active when bound to GTP, and in the absence of phosphorylation, catalyzes the translocation process necessary for protein elongation (Jorgensen, 2006).

24 12 Hydrolyzation of the inorganic phosphate on GTP yields GDP and provides the energy necessary for this process to occur (Proud, 2007). eef2 kinase is a calcium/calmodulin-dependent, regulatory kinase that controls eef2 activity by phosphorylating eef2 at Thr56 and thus inhibiting eef2 (Browne, 2002). With regard to translational elongation, mtor controls the phosphorylation and activity of eef2 kinase at three separate residues (Ser78, Ser359, Ser366) (Proud, 2007). Phosphorylation of eef2k on Ser78 is directly attributed to the hormone stimulated mtor pathway, but independent of p70s6k (Browne, 2004). eef2 kinase is phosphorylated (in vitro) by p70s6k on residue Ser366. This phosphorylation results in the inactivation of eef2 kinase (Wang, 2001; Browne, 2002). General inhibition of elongation occurs when eef2 kinase phosphorylates eef2 at Thr56 (Redpath, 1993). The addition of insulin has caused a decrease in eef2k activity, decline in eef2 phosphorylation to very low values immediately, and increased the transient time of elongation in vitro in Chinese hamster ovary cells. These resulting effects were suppressed following rapamycin treatment signifying a controlling effect over eef2k from the mtor pathway (Redpath, 1996). Acute resistance bouts in human participants also led to a decline in eef2 phosphorylation 1-2 hours post-exercise (Dreyer, 2010). Chronic overload in rats also results in a decline in signaling status (phosphorylation-to-total concentration) for eef2 as well (Thomson and Gordon, 2005). These data indicate that muscle overload likely affects elongation, in part by eef2 activation. The following sections will analyze how translational signaling proteins previously discussed are affected by muscle overloading and/or amino acid supplementation. Furthermore, the effects of aging on signaling responses to these stimuli will also be assessed.

25 13 Muscle Overloading and Resistance Exercise Skeletal muscle metabolism can be affected in a variety of ways when an external force (i.e. muscle overloading models and resistance exercise) is applied to the body. In order to quantify homeostatic responses in muscle protein synthesis and translational initiation and elongation signaling to these external forces, one must assess variables such as frequency (acute vs. chronic), duration (time), intensity (percentage of 1RM, VO2max, etc.), type and percentage of contraction (isometric vs. concentric vs. eccentric; maximal vs. submaximal, respectively), etc. This section will focus on differing modes of muscle overloading and resistance exercise in rodent and human subjects of various ages. The primary foci will be: 1) muscle protein synthesis rates, and 2) quantification of the phosphorylation status/total abundance of signaling markers associated with the Akt/mTOR pathway. Translational signaling markers found along the Akt/mTOR pathway have shown dramatic changes in response to acute resistance exercise. During 1- and 2- hours post-exercise, young adult participants increased in phosphorylation of Akt/PKB Ser473, mtor Ser2448, p70s6k Thr389, and decreased in phosphorylation for eef2 Thr56 (Dreyer, 2006). The pharmaceutical inhibitor rapamycin has been utilized to verify mtor s association with muscle protein synthesis in response to an acute bout of resistance exercise in young humans (Drummond, 2009). Increases in p70s6k Thr421/Ser424 and decreases in eef2 Thr56 phosphorylation attributed to resistance exercise were attenuated following rapamycin administration and corresponded with a ~40% reduction in muscle protein synthesis (Drummond, 2009). These data validated the correlation between skeletal muscle protein synthesis and mtor signaling. An important factor to consider was the possibility of gender differences in response to resistance exercise. Among male and female participants performing acute resistance exercise (10 sets of

26 14 10 reps at 70% 1RM), no variability was observed in mixed muscle protein synthesis (males, 52%; females, 47% increase at 2-hr post-exercise above baseline values). Furthermore, increases in phosphorylation of mtor Ser2448, p70s6k Thr389 and decreases in phosphorylation for eef2 Thr56 were not different at 1- and 2-hrs post-exercise between males and females, indicating that there were no acute gender differences among young humans (Dreyer, 2010). The incorporation of progressively increasing load volumes during acute resistance exercise has demonstrated variable effects on signaling protein phosphorylation and potential activation (Terzis, 2010). Additional sets of repetitions of leg presses revealed increased phosphorylation of p70s6k Thr389 and rps6 Ser235/236 at 30 minutes post-exercise for 3 and 5 sets of 6 maximum repetitions (3-fold, 5-fold; 30-fold, 55-fold, respectively)(terzis, 2010). Interestingly, no alterations occurred to the phosphorylation status of mtor Ser2448, possibly signifying that increasing load volume may not alter p70s6k and rps6 phosphorylation specifically through mtor activation. Acute resistance exercise also presented inconsistencies in the degree of phosphorylation for translational signaling markers between muscle fibers. Koopman et al (2006) demonstrated that type I and II muscle fibers responded to a bout of upper and lower body resistance exercise, with a larger increase in type II fibers for the phosphorylation of p70s6k Thr421/Ser424 within the first 30 minutes post-exercise (Koopman, 2006). During that time frame, rps6 showed no significant increases correlating with those of p70s6k; however, the phosphorylation of rps6 via mtor is controlled on by p70s6k Thr389 (Ruvinsky, 2005). Important to note were the effects that various types of muscle contractions had on protein synthesis and translational signaling. Maximal eccentric contractions demonstrated the most promising effects, with the greatest increases in phosphorylation of p70s6k Thr389, p70s6k Thr421/Ser424, and rp S6Ser235/236 between 1- and 2-hr post-exercise. Maximal concentric and

27 15 submaximal eccentric contractions had very little, if any, effect on acute signaling markers mentioned previously (Eliasson, 2006). These data suggested the additional possible involvement of a signaling pathway that bypass mtor when resisted contractions are presented as a stimulus. Muscle protein synthesis and translational signaling can also respond differently to different intensities and modes of exercise. Anaerobic cycling for 120 seconds at 110% of young male participant s VO 2 max resulted in an increased ratio of phosphorylated-to-total eef2 (2.3-fold) above rest. This response was accompanied by a shift in eef2. At rest, phosphorylation of eef2 was 55% greater in type I muscle fibers. Following exhaustive exercise, type II fibers exhibited 55% higher phosphorylation of eef2 (Rose, 2008). Recall that eef2 participates in the elongation stage of translation when dephosphorylated (Proud, 2007). With high intensities, type II fiber eef2 phosphorylation was suppressed immediately postexercise. However, acute low intensity, aerobic cycling (35%, 60%, and 85% of VO 2 max) did not show significant differences in elongation signaling (Rose, 2008). Previous research has been performed to analyze age- (Paturi, 2010) and gender-related (Smith, 2008) differences in phosphorylation and abundance of signaling markers at rest. Paturi et al (2010) measured resting phosphorylation and abundance status in extensor digitorum longus (EDL) muscles of adult, aged, and very aged male and female (6, 30, 36 month; 6, 26, 30 month, respectively) Fischer 344 x Brown Norway rats (Paturi, 2010). Values for Akt, mtor, and eef2 total abundance were higher (38%, 182%, 34%) and rps6 was lower (14%) in very aged rats when compared to adult rats. However, phosphorylation was significantly lower among signaling proteins downstream from mtor (p-p70s6k Thr389, 14%; p-rps6 Ser235/236, 21%; p-4e- BP1 Thr37/46, 24/25%; p-eef2 Thr56, 75%) in very aged rats compared to adult rats (Paturi, 2010). A comparison between very aged males and females revealed a significantly higher

28 16 phosphorylation status in all signaling markers previously mentioned for females at rest (Paturi, 2010). In a similar study among aged humans (65-80 yr), resting values for muscle protein synthesis and the phosphorylation status of specific signaling markers (p70s6k Thr389 and eef2 Thr56 ) favored fasted females (MPS, 30% greater, eef2 Thr56, ~40% less) (Smith, 2008). However, following the consumption of a standard meal (15% protein, 55% carbohydrate, 30% fat), aged males solely demonstrated an increase in muscle protein synthesis (and previously mentioned signaling markers) equivalent to the values observed in their female counterparts (Smith, 2008). These data suggested that a decline in phosphorylation, not expression, of signaling markers of the Akt/mTOR pathway existed during resting condition in aged rats, specifically in males (Paturi, 2010), and that feeding can restore protein synthesis in males to the level observed in aged-matched females (Smith, 2008). This lower level of resting muscle protein synthesis may account for the swifter progression of sarcopenia among males when compared to females (Janssen, 2000). Researchers have developed and implemented various overload models in rodents that mimic acute and chronic resistance training in humans. These methods have been utilized to study abundance and phosphorylation of translational signaling markers and muscle protein synthesis, as well as, to identify differences in responses to these stimuli between young and old rodents. High frequency electrical stimulation (HLES) is a common model of muscle overload performed in vitro. Adult (6 mo) and old (30 mo) Fischer344 x Brown Norway rats were subjected to a single session of 10 sets of 10 repetitions of HLES to determine the impact that acute in vitro muscle stimulation (via neuromuscular innervation) had on translational signaling markers mtor and p70s6k (Parkington, 2004). Of the muscles analyzed (tibialis anterior, TA; plantaris, PLT), neither showed age-related differences in mtor or p70s6k total abundance.

29 17 However, a muscle-specific (TA only) increase in mtor phosphorylation was reported with age (Parkington, 2004). Furthermore, an observed decline of 50% in post-hfes maximal phosphorylation of mtor and p70s6k occurred during the 6 hours following the end of stimulation in aged rats. It appeared that an acute post-stimulation diminishment exists in the ability to phosphorylate signaling markers (Parkington, 2004). Ablation of the synergist muscle(s) of the hind limbs of rats (i.e. gastrocnemious) is another commonly used model for chronic overload (Thomson and Gordon, 2005; Thomson and Gordon, 2006; Chale-Rush, 2009). Chale-Rush et al incorporated a bilateral synergist ablation or sham surgery on young (6 mo) and old (33 mo) Fischer 344 x Brown Norway rats for 28 days. Following normalization for body weight, the overloaded plantaris muscles of young and old rats that underwent synergist ablation were 35% and 20% heavier than the plantaris muscles of control rats. Despite increases in muscle weight, a 15% greater amount of hypertrophy was achieved in the young rats when compared to the old. At the end of the study (28 days), expression of mtor, p70s6k, rps6 and 4E-BP1 was unaffected by age or overload. However, mtor and rps6 phosphorylation was greater (44%, 35%; 114%, 24%) in overloaded young and old plantaris muscles over the controls, respectively (Chale-Rush, 2009). Interestingly, agerelated differences in signaling protein phosphorylation within the overloaded group were no longer significant after 28 days (significant differences were present after 7 days) (Chale-Rush, 2009). In a similar study, young adult (8 mo) and old (30 mo) Fischer 344 x Brown Norway rats underwent 1-week unilateral synergist ablation of the gastrocnemious muscle to determine the effects of aging on muscle hypertrophy (Thomson and Gordon, 2005). Again, increased muscle wet weight for both young adult and old rats were above that of the intra-specimen sham

30 18 (control) muscles. Nevertheless, old rat plantaris muscles still exhibited hypertrophy to a lesser extent when compared to control muscles than did their younger counterparts (Thomson and Gordon, 2005). After only 7 days of overload, mtor phosphorylation was substantially higher in young vs. old adults (292% vs, 88%, respectively) (Thomson and Gordon, 2005). Thomson and Gordon utilized the same experimental protocol previously discussed (Thomson and Gordon, 2005) to quantify the phosphorylation-to-total concentrations (labeled signaling status) of signaling proteins Akt, mtor, p70s6k, rps6, eef2, and 4E-BP1 in young and old rats (Thomson and Gordon, 2006). Signaling statuses for mtor, p70s6k, rps6 and 4E-BP1 showed increases in both young adult and old overloaded rats when compared to their age matched controls. Also, Akt (old rats only) and eef2 statuses showed marked decreases. Important to note is that these increased and decreased phosphorylation statuses were significantly greater in young adult rats, indicating that there was a signaling deficit in with age in response to muscle overload. Additionally, a correlation between absolute p70s6k phosphorylation and muscle hypertrophy was established from this study (Thomson and Gordon, 2006). When the duration of muscle overloading was increased, Blough et al observed greater gains in contractility, whole muscle fiber (plantaris) and muscle fiber cross sectional area (CSA) over an 8-week time period in adult (8.5 month) rats over the age matched controls (Blough, 2000). On the other hand, overload alone was not able to rescue the hypertrophic response of whole plantaris muscle or individual muscle fiber cross-sectional areas in old (38 month) rats, and actually showed declines in the CSA of type I, IIA, and IIX/IIB (31, 35, 39%) muscle fibers, respectively (Blough, 2000). There is an apparent inability to stimulate muscle protein synthesis and the phosphorylation of specific translational signaling markers in old rats to the extent produced in young/adult rats (Parkington, 2004; Thomson and Gordon, 2005, Thomson and Gordon, 2006; Chale-Rush, 2009). Whether

31 19 the deficiencies observed in these elements solely contribute to the inability to stimulate an anabolic response, or whether they work in conjunction with other mechanisms, has yet to be determined. In terms of exercise intensity, different acute dosages can cause variance in signaling marker phosphorylation in humans, especially when age is a variable. Twenty-five young and 25 old fasted men performed an acute bout of leg extension and flexion exercise at intensities ranging from 20% to 90% of the participant s 1RM with corresponding sets and repetitions (i.e. 20% 1RM, 3 sets x 27 reps; 75% 1RM, 3 sets x 8 reps; etc.) (Kumar, 2009). A relationship was established between the intensity of resistance training and myofibrillar muscle protein synthesis (MPS), where gradual increases were generated from 20% to 60%, followed by a plateau at 75%. Gains in MPS were 30% greater among young subjects than in old (Kumar, 2009). Maximal phosphorylation of 4E-BP1 and p70s6k was observed during 1-hr post-exercise and these increases were correlated with the increase in MPS, although only among young participants. eef2 phosphorylation was unaffected by any variability in age or intensity (Kumar, 2009). It was apparent that acute in vivo stimulation of MPS and the correlating phosphorylation of muscle protein translation initiation proteins were depressed or blunted in aged humans. Chronic resistance training has demonstrated variability in its stimulatory effects on muscle hypertrophy amongst young and old humans. Kosek et al (2006) examined the effects of a chronic 16-weeks resistance-training regimen performed 3 days per week using 3 knee extensor exercises at 3 sets for 8-12 reps per set (Kosek, 2006). The chronic cycle of resistance training generated increases in type I (18%) and II (32%) fiber cross-sectional area (CSA) among young males and females (20-35 yrs). Type I fibers showed no increases in CSA, while type II fibers exhibited limited increases (23%) in CSA among older subjects (60-75 yrs) (Kosek, 2006).

32 20 Furthermore, a transition from type IIx fibers to type IIa fibers was observed in all groups (females 47.4+/-1/7 to 60+/-2.3%; and males /-2.3 to /- 2.5%). One positive result is that older individuals were able to stimulate growth in type II fiber CSA after 16-weeks of exercise to the baseline level of younger subjects indicating that there is the potential for muscle growth. However, the hypertrophic response was still not as dramatic as that observed in younger humans at the end of the training period (Kosek, 2006). These data contradict those of Blough, 2000 stating that declines were actually observed in muscle fiber CSA follow 8-weeks of muscle overloading in aged rats (Blough, 2000). Longer duration of resistance training and variability in the mode used to cause physical stress on the muscles may account for these differences. From the data discussed, it is clear that aging produces a desensitization to muscle overloading as a stimulus for skeletal muscle hypertrophy and translational signaling. Researchers have examined nutritional supplementation as a possible intervention for sarcopenia. The findings will be discussed at length in the following section. Supplementation of Whey Protein and Amino Acids Nutritional supplementation has been shown to act as a catalyst by enhancing muscle protein synthesis above the stimulatory effects observed in the fasted state (Burd, 2010). Recent focus has been centered around the addition of dietary whey protein (Paddon-Jones, 2006; Rieu, 2006; Burd, 2010,) and the amino acids that comprise this ergogenic aid (essential and nonessential amino acids)(dardevet, 2000; Dardevet, 2002; Guillet, 2003, Paddon-Jones, 2004; Katsanos, 2006; Paddon-Jones, 2006; Fujita, 2007; Atherton, 2010; Katsanos, 2008; Dickinson, 2011) in an attempt to determine which specific component(s) contributed to the stimulatory effect on muscle protein synthesis.

33 21 An instrumental experiment performed by Volpi et al quantified protein synthesis and degradation kinetics among differing age groups (Volpi, 2001). Kinetic markers for protein synthesis and breakdown were examined in the vastus lateralis of fasted young (28+/-2yr) and elderly (70+/-1yr) male humans without dietary manipulation. Among participants, net muscle protein balance was equal. Remarkably, muscle protein synthesis was higher in elderly males. Researchers speculated that equal values in net protein balance, rather than values favoring the elderly group, were a product of the equally high degradation levels among elderly participants (Volpi, 2001). In a separate study, gender differences where quantified in the fasted- and fedstates among aged humans (Smith, 2008). Aged females in the postabsorptive state exhibit a greater percentage of basal (fasted) muscle protein synthesis (~30%) than those values observed in aged males (Smith, 2008). These differences are not observed in p70s6k Thr389 phosphorylation at basal levels. Following the consumption of 15 small liquid meals (15% protein, 55% carbohydrate, 30% fat) over 150 minutes, increases in p70s6k phosphorylation occurred to the same extent in both gender groups. However, increases in MPS were only found to exist in males (Smith, 2008). From these data, it was evident that the consumption of a meal acted to increase muscle protein synthesis and exhibited greater values for muscle protein synthesis measured in the fasting state. It is important determine whether one, or a combination of components, found in a standard meal generated these observed increases in muscle protein synthesis and translational signaling. Proteins are comprised of amino acids, which are classified as either essential (EAA) or non-essential amino acids (NEAA), and can be supplemented together (i.e. in whey protein) or separately (Katsanos, 2008). Amino acids administered via intravenous infusion to young human participants in the postabsorptive state have demonstrated an initial delay or latency

34 22 period of ~30 minutes, followed by a dramatic increase (2.8-fold; 0.08+/ (basal) to 0.21+/ (30-60min) to 0.24+/-0.04%/hr ( min)) in mixed muscle protein synthesis between 30 and 120 min (Bohe, 2001). These data suggested that amino acids administered in vivo could facilitate increases in muscle protein synthesis, although there was a delay in the initiation of protein synthesis. To further discern which specific component(s) possess anabolic properties, amino acids were separated into their respective EAA and NEAA groups and measured in vitro and in vivo in order to determine their anabolic properties on protein synthesis and accrual (Atherton, 2010; Katsanos, 2008). Thirty minutes of in vitro incubation of specific amino acids to C2C12 myocytes revealed that neither a combination of nonessential amino acids (NEAA), nor the branched-chain amino acids (BCAA) valine or isoleucine, acted as stimuli for phosphorylating initiation signaling markers and muscle protein synthesis (Atherton, 2010). On the contrary, in vitro incubation with essential amino acids (EAA) and leucine independently caused dramatic increases in the phosphorylation of mtor Ser2448, 4E- BP1Thr37/46, rps6 Thr235/236 and especially p70s6k Thr389 (p-p70s6k: EAA, fold increase; leucine, 5.9+/-0.5 fold increase) (Atherton, 2010). Administration of NEAA (7.57g) in vivo has shown no affect on protein accumulation at 3.5 hr post-ingestion among elderly humans (Katsanos, 2008). Although EAA (6.72 g) demonstrated an increase in protein accrual, these values could not compare to the increases observed when whey protein was supplemented (15 g) (Katsanos, 2008). The exact signaling pathway(s) by which EAA elicits such effects remains unclear, but stimulation of either mtor pathway signaling marker phosphorylation or muscle protein synthesis has been observed following the administration of EAA (Atherton, 2010). Cellular transport allows for essential components of protein synthesis, such as amino acids, to cross the cell membrane and become incorporated in the production of proteins. Skeletal muscle amino

35 23 acid transporters LAT1/CD98 and SNAT2 have been associated with acute mtor pathway stimulation (via phosphorylation of rps6 Ser240/244 ) at 1-hr post-administration of 10 g of a mixture of essential amino acids (Drummond, 2010). Acute resistance exercise has caused equivalent expression and upregulation of the fore-mentioned transporters along with others (LAT1/SLC7A5, SNAT2/SLC38A2, CD98/SLC3A2, PAT1/SLC36A1, and CAT1/SLC7A1), in young (28+/-2yr) and old (68+/-2 yr) humans (Drummond, 2011). Although neither expression nor upregulation of these amino acid transporters was significantly different between age group, mtor associated stimulation previously observed (Drummond, 2010) in the form of phosphorylation of rps6 Ser240/244 was substantially lower in older humans (Drummond, 2011). Desensitivity to amino acid stimulation with age (Paddon-Jones, 2004) could be associated with cellular transporters lacking the ability excite activity associated with translational signaling and muscle protein synthesis. For that reason, we chose to also evaluate the effectiveness of nutrients (specifically, the amino acid leucine) on stimulating increases in phosphorylation and abundance of downstream mtor signaling markers, as well as muscle hypertrophy. Lack of excitation by nutrients, combined with a reduced ability of muscle overload/resistance exercise to generate anabolic responses in translational signaling and muscle protein synthesis in old rats and humans, equivalent to that of young, could indeed be the major cause for the decrements in muscle mass and quality with age. A key study by Dickinson et al also helped to provide clarity on this issue. The specificity of EAA on mtor signaling was demonstrated in fasted young human participants following an acute administration of an essential amino acid solution (Dickinson, 2011). As expected, muscle protein synthesis and phosphorylation of mtor Ser2448, 4E- BP1Thr37/46, and p70s6k Thr389 increased dramatically at 1-hr post-administration. These effects on muscle protein synthesis and the